Overview
This lecture covers amino acids, peptides, and proteins, including their structure, classification, properties, and methods for protein purification and analysis.
Amino Acids: Structure & Isomerism
- Amino acids have a central (alpha) carbon: bonded to an amino group, carboxyl group, hydrogen, and side chain (R group).
- All amino acids except glycine are chiral and exist as two enantiomers (L and D).
- Proteins are made from L-amino acids.
- Enantiomers differ in spatial arrangement and interact differently with polarized light (optically active).
Amino Acid Classification & Properties
- Five main classes: nonpolar aliphatic, aromatic, polar uncharged, positively charged, negatively charged.
- Nonpolar and aromatic amino acids contribute to the hydrophobic effect, stabilizing protein structure.
- Polar uncharged groups form hydrogen bonds; cysteine forms disulfide bonds.
- Charged groups affect protein structure and are relevant at physiological pH.
Acid-Base Properties & Titration
- Amino acids can act as acids, bases, or zwitterions (net zero charge at isoelectric point, pI).
- Standard amino acids have two ionizable groups (amino and carboxyl); some have an ionizable side chain (third group).
- The isoelectric point is calculated as the average of pKa1 and pKa2 if only two groups are ionizable.
- At pH = pI, amino acids have zero net charge and minimal solubility.
Peptides and Proteins
- Peptide bonds form between amino acids via condensation (loss of water).
- Peptides: chains of amino acids; proteins: long polypeptides (>10 kDa).
- Sequence is read from N-terminal (amino) to C-terminal (carboxyl).
- Learn both three-letter and one-letter amino acid codes.
Protein Purification and Characterization
- Proteins can be separated by size, charge, or binding properties.
- Chromatography types: ion exchange (charge), size exclusion (size), affinity (binding specificity), HPLC (high-pressure).
- Electrophoresis separates proteins by charge/mass ratio; SDS-PAGE uses detergent to unfold proteins for separation by size.
- Protein molecular weight is estimated by comparison to standards in gels.
Protein Structure and Sequencing
- Four structural levels: primary (sequence), secondary (alpha helices/beta sheets), tertiary (3D shape), quaternary (multiple subunits).
- Amino acid sequence determines protein structure and function.
- Sequencing methods: Edman degradation (chemically removes N-terminal residues), protease digestion, and mass spectrometry.
Protein Synthesis and Evolution
- Small proteins can be synthesized chemically using protecting groups and stepwise addition.
- Sequence data inform structure, function, localization, and evolutionary relationships.
- Homologs: proteins of the same family; paralogs: within-species homologs; orthologs: homologs across species.
- Consensus sequences highlight conserved functional residues.
Key Terms & Definitions
- Amino Acid — organic molecule with amino, carboxyl, hydrogen, and variable side chain (R group).
- Enantiomer — mirror-image isomer, optically active.
- Zwitterion — molecule with both positive and negative charges but net zero charge.
- Isoelectric Point (pI) — pH at which a molecule has zero net charge.
- Peptide Bond — covalent bond linking amino acids in peptides/proteins.
- Protease — enzyme that hydrolyzes peptide bonds.
- SDS-PAGE — electrophoresis method using SDS detergent for protein separation by size.
Action Items / Next Steps
- Memorize amino acid structures, names, and one- and three-letter codes.
- Review acid-base and titration curve principles for amino acids.
- Practice drawing peptide structures and calculating net charge at different pH values.
- Prepare for practice exercises on protein purification and sequence analysis.